Chromosome analysis is an important component in diagnosing congenital anomalies that cause physical and mental developmental delay. Cytogenetic imbalance results in DNA copy-number changes and alteration in gene dosage in the altered chromosomal segment(s). These changes may result in abnormal clinical phenotypes. Such chromosomal aberrations are conventionally detected by a variety of methods, each with distinct advantages and disadvantages. Routine cytogenetic analysis by GTG banding can achieve a resolution sufficient to detect aneuploidy and structural rearrangements of base-pair sequences greater than five megabases (Mb) but cannot reliably identify abnormalities less than five Mb.
More subtle genetic alterations or those involving regions that are difficult to visualize may be undetectable by conventional cytogenetic techniques (e.g., these include most microdeletion syndromes and exchanges of similarly banded segments that lead to cryptic translocations). Fluorescence in situ hybridization (FISH) was developed to probe individual chromosomal loci at a resolution equal to the size of the probe (e.g., 35-200 kilobases (Kb)). Only a few loci may be examined at a time, however, and FISH can usually only be performed in a limited manner based on phenotype. Thus, single locus FISH is not an appropriate screening tool for the analysis of more than a few loci at a time.
Additional molecular cytogenetic techniques were developed to overcome these limitations. Comparative genomic hybridization (CGH) was developed to identify chromosomal imbalance without the need for phenotypic information, circumventing multiple FISH experiments. CGH provides genome-wide screening of genetic sequence alterations by comparing differentially labeled test and control samples of genomic DNA. The resolution of the technique, however, is still limited to approximately 5-10 Mb because metaphase chromosomes are used as the targets for analysis.
To substantially increase the resolution, CGH-based microarrays for performing “array CGH” were developed. Array CGH is a high-resolution, comprehensive method for detecting both genome-wide and chromosome-specific copy-number imbalances. Array CGH typically uses large-insert clones (such as bacterial artificial chromosomes, “BACs”) as the target for analysis rather than metaphase chromosomes. As a consequence, the resolution of the array is limited only by the size of the insert used and the physical distance in the human genome between clones that are selected for the array.
CGH microarrays have been successfully constructed to test many parts of the human genome. In 2001, a whole-genome array was constructed using approximately 2400 BAC clones to scan for genome-wide copy-number alterations. An array covering some of the telomeric regions of the human genome has also been developed. Individual chromosomal regions have also provided good targets for array CGH. For example, in 2003 a microarray was designed to cover much of the most distal 10.5 Mb of chromosome location 1p36 to study subjects with monosomy 1p36. In 2003, an array was constructed based on chromosome 18 to study patients with congenital anal atresia. Microarrays have also been developed to test parts of chromosomes 20 and 22.
As shown in FIG. 1, conventional CGH microarrays 100 constructed for research purposes are designed to screen chromosomal regions or the entire genome for chromosomal segment gains or segment losses with improved resolution over earlier techniques. For many reasons, however, most of these are not appropriate or relevant for use in clinical genetic diagnosis. First, most conventional BACs used in these microarrays have been culled from BAC databases without prior external verification of the exact locations (“loci”) that they map to (“cover”) on a chromosome 102. Second, these databases rarely provide notice that some of the BACs map to multiple loci, even on entirely different chromosomes 104. Third, many of the loci that are covered by a single clone may show dosage variation due to the inherent technical variability of the procedures involved 106. Fourth, the conventional CGH microarray 100 may identify alterations in regions of the genome that do not have established clinical relevance 108. Thus, such conventional “whole genome” arrays are likely to generate data that are difficult to interpret or that are inaccurate in that they present multiple false positive results 110. That is, alterations in regions of the genome that do not have established clinical relevance are impossible or, at the very least, expensive to interpret and/or verify in a clinical setting 112.
In a clinical setting, a conventional whole-genome approach to array CGH may cause erroneous test results that result from undesirable polymorphisms, which are usually abundantly represented in this approach. Data from sub-telomere FISH analysis, for example, reveal many telomeric alterations that possess no clinical significance.
It is estimated that about 35% of clones that are currently available from the public and private databases either map to the wrong location, map to more than one location in the genome, represent polymorphic areas of the genome, or contain repetitive sequences that may interfere with hybridization. Using random clones from the databases would result in a clinical test that has more than a 35% probability of error and that is of dubious utility. Thus, “whole genome arrays” arrays are not appropriate for clinical applications. The adoption of such “whole genome” arrays for use in clinical diagnostics may be unwise, not only leading to many false positive diagnoses that necessitate expensive follow-up confirmatory tests by FISH or other methods (e.g., 112); but also additional blood draws from unaffected relatives of the patient to determine possible segregation of genetic deletions, duplications, or polymorphisms; not to mention unnecessary anxiety for the family of a person being tested. In the “whole genome” approach, hybridization results for single clones that show dosage difference require careful examination and each clinical case may require all the time and expense of a mini-research project. Thus, genome-wide “dense” arrays that are conventionally available for research use are not appropriate, relevant, or efficient in a clinical setting. There exists a need for a clinically useful diagnostic array that provides reliability, that accurately detects chromosome abnormalities assayed, and that provides interpretable results with an acceptable degree of precision. Further, there exists a need for methods of precision genetic diagnosis that provide clinical confidence by interrogating clinically relevant parts of the genome rather than the clinically irrelevant parts.